DefinePK

Abstract

An accurate sexing process is crucial for preserving the purity of Pesisir cattle bulls and enhancing their productivity to meet the increasing beef demand in Indonesia. Therefore, this study aims to verify the separation of spermatozoa carrying X and Y chromosomes molecularly, using the Bovine Serum Albumin (BSA) column. The spin-column method was employed to isolate the separated X and Y spermatozoa. Amplification through PCR was performed using two sets of primers targeting the Sex-determining Region Y (SRY) gene, located on the Y chromosome, and the Ausal Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene on the X chromosome. The assessment of spermatozoa quality in coastal cattle bulls showed an average fresh semen volume, and sperm concentration to be 3±0.7mL, and 1.58±1.05×106/mL, respectively. While the other parameters of assessment of spermatozoa quality including average motility, spermatozoa survival, sperm abnormalities, and mean progressive motility (MPU) were to be 80±10, 85.83±7.59, 6.36±3.50, and 79.81±5.97%, respectively. The quality evaluation after sexing included the average motility of spermatozoa with X at 56.66±11.54 and Y at 66.66±11.54%, as well as their average intact plasma membrane (IPM) being 55.58±1.01 and 59.35±7.78%, respectively. The quality of spermatozoa decreased by 20-30% after sexing. The results of spermatozoa separation with the 5% BSA column confirmed one GAPDH band (415bp), indicating the content to be X. In contrast, the 10% BSA column and the non-sexed spermatozoa exhibited two bands, SRY (318bp) and GAPDH (415bp), indicating a higher proportion of Y in the 10% BSA column. These demonstrated molecular verification of sexed spermatozoa using the 5 and 10% BSA column, enabling the separation of those carrying X and Y chromosomes in Pesisir bulls through the duplex PCR method.